Ghrelin, an endogenous growth hormone secretagogue (GHS) to growth hormone secretagogue receptor (GHS-R) which is one of orphan receptors, is a physiologically active peptide first isolated and purified from rat in 1999 (Kojima, et al., Nature, 402: 656-660, 1999). Thereafter, some ghrelins having same chemical structure of rat ghrelin have been isolated from vertebrates other than rat, such as human, mouse, pig, chicken, eel, bovine, equine, ovine, frog, trout and canine. The chemical structures of these ghrelins are listed in the following Table 1.
TABLE 1HumanGSS(n-octanoyl)FLSPEHQRVQQRKESKKPPAKLQPRGSS(n-octanoyl)FLSPEHQRVQRKESKKPPAKLQPRRatGSS(n-octanoyl)FLSPEHQKAQQRKESKKPPAKLQPRGSS(n-octanoyl)FLSPEHQKAQRKESKKPPAKLQPRMouseGSS(n-octanoyl)FLSPEHQKAQQRKESKKPPAKLQPRPorcineGSS(n-octanoyl)FLSPEHQKVQQRKESKKPAAKLKPRBovineGSS(n-octanoyl)FLSPEHQKLQRKEAKKPSGRLKPROvineGSS(n-octanoyl)FLSPEHQKLQRKEPKKPSGRLKPRCanineGSS(n-octanoyl)FLSPEHQKLQQRKESKKPPAKLQPREelGSS(n-octanoyl)FLSPSQRPQGKDKKPPRV-NH2TroutGSS(n-octanoyl)FLSPSQKPQVRQGKGKPPRV-NH2GSS(n-octanoyl)FLSPSQKPQGKGKPPRV-NH2ChickenGSS(n-octanoyl)FLSPTYKNIQQQKGTRKPTARGSS(n-octanoyl)FLSPTYKNIQQQKDTRKPTARGSS(n-octanoyl)FLSPTYKNIQQQKDTRKPTARLHBullfrogGLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNMGLT(n-decanoyl)FLSPADMQKIAERQSQNKLRHGNMGLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNMNTilapiaGSS(n-octanoyl)FLSPSQKPQNKVKSSRI-NH2CatfishGSS(n-octanoyl)FLSPTQKPQNRGDRKPPRV-NH2GSS(n-octanoyl)FLSPTQKPQNRGDRKPPRVGEquineGSS(n-butanoyl)FLSPEHHKVQHRKESKKPPAKLKPR(wherein, an amino acid residue is written by the one letter notation defined by IUPAC and IUC)
These peptides are characterized by a specific structure due to acylation of hydroxyl group at the side chain of serine group (S) or threonine group (T) by fatty acid such as octanoic acid or decanoic acid, and there has never been isolated the physiologically active peptides having modifying hydrophobic group such as ghrelin. These new peptides exhibit potent promoting effect for secretion of growth hormone, and it has become clear that these peptides perform for adjusting the secretion of growth hormone. Therefore, many researchers have great interest in physiologically active roll of ghrelin and for development of these peptides as medicines (e.g., World Patent Publication WO 01/07475).
It is known that the modifying hydrophobic group in ghrelin molecule has to be necessary for exhibiting the physiological effects (Kojima, et al., Nature, 402; 656-660, 1999). However, due to the non-existence of peptides like ghrelin having the modifying hydrophobic group in molecule at the hydroxyl group of side chain of specific amino acid residue, the stability of these peptides for development as medicines have never been studied.
Incidentally, the compound to be developed as medicines has the various kinds of chemical structures, and because of these chemical structures, the compounds may easily degrade in the formulation process or in the storage process thereafter. The degradation reactions are hydrolytic cleavage, dehydration, isomerization, oxidization, reduction or photodegradation of the compound, and further, the chemical reaction of the compound with additives to be formulated with the compound. Therefore, it is very important to study and understand the varieties of the degradation reaction and the degrees thereof from the chemical structure of the compounds, for development of the compound as medicines, and consequence quality control thereof.
It is well known that the stability of medicines may be greatly controlled by the ambient environmental condition, such as pH level of the environment. The influence of pH of the solution for the degradation rate of medicines in aqueous state has been studied, and pH profile of degradation rate of many medicines has been reported (e.g., Sumie Yoshioka, “Stability of Medicines” by Nankohdo, 1995).
The physiologically active peptides or physiologically active proteins are inactivated and degraded by protease existing in the digestive organ, and it is difficult to develop the oral administrable composition containing these peptides or proteins. Therefore, these peptides or proteins are prepared as an injectable composition for the clinical administration, and for this purpose the stability of these substances in the aqueous solution is very important for preparation of the liquid pharmaceutical formulations regardless of the dosage form such as solution form or soluble solution form in site.
At present, pharmaceutical compositions containing various kinds of peptide or protein such as insulin, growth hormone, calcitonin, atrial natriuretic peptide, LH-RH (luteinizing hormone-releasing hormone) derivatives or adrenocorticotropic hormone derivatives are on sale as medicines, and it is reported that the chemical changes of these peptides or proteins are deamidation, iso-aspartic acid formation, hydrolytic cleavage such as fragmentation, racemization, formation of disulfide bond or exchange reaction, β-elimination or oxidative reaction.
These chemical changes exert an influence on the stability of the composition containing peptides or proteins, and the degrees of the degradation reaction of peptides or proteins is dependent on a pH value of the solution. For example, it is reported that the chemical structure of degradation products and the produced amount of the degradation products varied according to the pH value of the solution containing these peptides or proteins, such as LH-RH derivatives (Strickley et al., Pharm. Res., 7, 530-536, 1990), human parathyroid hormone (Nobuchi et al., Pharm. Res., 14, 1685-1690, 1997) hirudin (antithrombin substance: Gietz et al., Pharm. Res., 15, 1456-1462, 1998), and human amylin derivatives (Hekmann et al., Pharm. Res., 15, 650-659 1998).
Ghrelin or its derivative of the present invention is a physiologically active peptide, and it is common to prepare an aqueous solution containing ghrelin as pharmaceutical composition for medicine. Though the stability of ghrelin in the aqueous solution is very important for preparation of the pharmaceutical composition, there has never been any study of the stability of ghrelin in the aqueous solution. Ghrelin or its derivative has the specific modifying hydrophobic group in its molecule, that is, the side chained hydroxyl group of certain amino acid residue of ghrelin or its derivative is acylated by fatty acid. There has never been discovered a peptide like ghrelin having the specific modifying hydrophobic group in molecule, therefore, the common knowledge about the stability of ghrelin has also never been reported. That is, it is unknown about the stability, the chemical structure of degradation product and the mechanism of production of the degradation product of ghrelin. Further, it is unknown about the mechanism of degradation of modifying hydrophobic group of ghrelin, as well as the secondary degradation from the degradation product of ghrelin.
Under these circumstances, the objective of the present invention is to provide a pharmaceutical composition stably containing ghrelin or its derivative and a method for preventing degradation of modifying hydrophobic group of ghrelin or its derivative in an aqueous solution dissolved ghrelin or its derivative therein based on the knowledge obtained by the investigation of the chemical stability of ghrelin or its derivative having specific modifying hydrophobic group in the molecule.
Through extensive investigations of the influence of pH in an aqueous solution containing ghrelin and the chemical structure of the degradation product from ghrelin, the present inventors discovered that, in an aqueous solution, ghrelin degraded to produce desacyl compound by hydrolytic cleavage of the specific modifying hydrophobic group and in addition, degraded to produce dehydroalanine compound by β-elimination of modifying hydrophobic group, consequently to produce the secondary degradation product due to the volatility of dehydroalanine compound, and these degradation were affected by pH value of the aqueous solution.
Based on the results of the mechanism of degradation of ghrelin mentioned above, the present inventors further discovered that the pharmaceutical composition stably containing ghrelin could be obtained by adjusting pH of the solution with pH adjuster or buffer agent, and this stabilization effect could be obtained by various sorts of the buffer agent independent of their concentration or ghrelin concentration, and thus completed the present invention.